AB0035 CHEMOKINES (IL-8 &amp; GRO-Α) AND CXCR1/2 PATHWAY INDUCES SENESCENCE INDUCTION IN CHONDROCYTES
Authors: A. K. Anuja, A. K. Srivastav, M. K. Rai, L. Gupta, D. P. Misra, V. Agarwal
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Tumor Necrosis Factor-α(TNF- α) and Interleukin(IL-1) are well-known in the induction of senescence in chondrocytes and play an important role in the pathogenesis of Osteoarthritis (OA). However, targeting these pathways has not yielded promising results. The chemokines IL-8(CXCL8) and Gro-α(CXCL1), as well as the CXCR1/2 pathways, are known to mediate inflammation and apoptosis. However, the precise role of these chemokines in the pathogenesis of OA is unknown. We hypothesize that the CXCR1/2 signaling pathway is involved in the induction of senescence in chondrocytes. <h3>Objectives</h3> To evaluate the role of chemokines and CXCR1/2 pathway in induction of senescence in chondrocytes derived from patients with OA and healthy chondrocytes cell. <h3>Methods</h3> Healthy primary chondrocytes(HPC) cell lines and human primary chondrocyte cells derived from patients diagnosed with OA cultured and expanded up to passage 5. Cells were induced with IL-8 and GRO–a followed by determining the senescence marker via β -galactosidase(β-GAL, histochemical staining) and UPAR and P16NKa using qRT-PCR. Apoptosis in both HPCs and OPCs were done via Annexin V and Proprium iodide (PI) using Flow-cytometry. Statistical analysis was performed using un-paired t test and One-way-Anova. <h3>Results</h3> This study showed that the % of β-GAL in the HPC were almost negligible. However, showed significantly higher % in OPC(<i>p&lt;0.001</i>). Moreover, when stimulated with IL-8, exhibited markedly increase in the positive blue cells in both HPCs and OPCs in a time dependent manner (p&lt;<i>0.001</i>)(at 24 hrs., 48 hrs., 72 hrs. &amp; 96 hrs.). In line with results of HPC and OPC stimulated with IL-8, OPC also showed higher % of senescence when induced with Gro- α (p&lt;<i>0.001</i>)(at 24 hrs., 48 hrs.,72 hrs. &amp; 96 hrs <b>{Figure 1(A)(B)(C)}.</b> Since TNF-α and IL1-β reported to have role in induction of senescence in chondrocytes, we have examined the β-GAL in HPC and OPC treated with TNF-α and IL1-β and found significant higher no. of blue stained in the OPC<b>(Table 1</b>). These results were further validated by fold change expression of senescence induced genes <i>P16NKa</i> and <i>UPAR</i> at time dependent manner. We observed higher fold change expression of <i>P16NKa</i> in IL-8 induced OPC at 24 hrs. (<i>p=0.01</i>); at 48 hrs. (<i>p&lt;0.001</i>); at 72 hrs. (<i>p=0.03</i>) and at 96 hrs. (<i>p&lt;0.001</i>) with that of IL-8 induced HPC. However, stimulation with Gro-α showed increase in the expression of <i>P16NKa</i> in a time dependent manner(at 24 hrs., 48hrs., 72hrs. and 96 hrs.) in both OPCs and HPCs. Similarly, the expression of <i>UPAR in</i> IL-8 induced OPCs at 24 hrs.(<i>p=0.69</i>); at 48 hrs.(<i>p=0.05</i>); at 72 hrs. (<i>p&lt;0.0001</i>) and at 96 hrs.(<i>p=0.01</i>) with that of Gro–α induced HPC. While the expression of <i>UPAR</i> in Gro–α induced OPCs and HPCs showed increase in a time dependent manner(at 24 hrs., 48 hrs., 72 hrs. and 96 hrs.). Flow-cytometric analysis revealed that induced cells were negative expression for apoptotic markers viz., Annexin V and PI. <h3>Conclusion</h3> Overall, this study highlights that IL-8 and Gro- α induce senescence in chondrocytes, a key event in the pathogenesis of OA. However, CXCR2 pathway may be explored further for therapeutic potential. <h3>Reference</h3> [1]Cecil DL, Rose DM, Terkeltaub R, Liu-Bryan R. Role of interleukin-8 in PiT-1 expression and CXCR1-mediated inorganic phosphate uptake in chondrocytes. Arthritis &amp; Rheumatism: Official Journal of the American College of Rheumatology. 2005 Jan;52(1):144-54. <h3>Acknowledgements</h3> Anamika Kumari Anuja acknowledges Department of Biotechnology, Ministry of Science and Technology, Government of India for her DBT -JRF/SRF -programme fellowship. Intramural Research Grant, SGPGIMS, Lucknow. <h3>Disclosure of Interests</h3> None Declared.